Innate immune detection of nucleic acids is essential for protective immunity against viral infection, but can cause autoimmune disease if dysregulated. This application is based on the study of an antiviral response triggered by detection of intracellular DNA called the interferon stimulatory DNA (ISD) pathway. Despite the established importance of DNA sensing in immunity and certain human autoimmune diseases, the identities of the DNA receptors that activate these responses remains controversial. In particular, AIM2-like receptors (ALRs) and cyclic GMP-AMP synthase (cGAS) have emerged as attractive candidate DNA sensors, but the relative contributions of these receptors to immunity, alone and together, remain to be established. We developed novel tools to explore the functions of ALRs, cGAS, and other candidate DNA sensors: mice that lack all thirteen ALR genes, and knockout human cell lines lacking candidate DNA sensors. We will use these exciting new tools to address the following specific aims. Aim 1: What are the relative roles of murine ALRs and cGAS in intracellular DNA sensing? Using cells from mice that lack all ALRs, cGAS, or both ALRs and cGAS, we will perform a comprehensive evaluation of DNA-activated innate immune responses to pure DNA ligands, as well as to infections with DNA viruses and intracellular bacteria. Aim 2: What are the in vivo roles of candidate DNA sensors in host defense and autoimmune disease? We will establish the relative contributions of ALRs and cGAS to activation of immunity in vivo using a plasmid DNA-based vaccination approach. We will then determine the roles of ALRs and cGAS in a mouse model of autoimmune disease caused by chronic activation of the ISD pathway. Aim 3: What are the functions of candidate DNA sensors in human cells? We have used CRISPR-mediated genome engineering to achieve biallelic disruption of genes in the relevant human monocyte cell line THP-1. We will establish panels of gene-targeted THP-1 cells for a rigorous analysis of the roles of candidate DNA sensors in activation of the ISD pathway and the inflammasome in response to pure ligands, and during infection with DNA viruses and lentiviruses that have exclusive tropism for human cells. Together, these proposed studies will enable us to explore in detail the mechanisms of intracellular DNA sensing in both mouse and human cells.